Squamous cell carcinoma of the head and neck (SCCHN) is the sixth most common cancer worldwide [1]. The overall survival rate for SCCHN of approximately 50% in five years has not changed very much in recent decades, so there is a major need for new approaches for the prevention and treatment of SCCHN. With funding from this grant, we developed a novel mouse model of oral cavity carcinogenesis, the 4-nitroquinoline-1 oxide (4-NQO) drinking water model [2]. We used this model to establish that RA (all-trans retinoic acid) and/or 5-aza-dCdR (the DNA methyltransferase (DNMTase) inhibitor 5-aza-2'-deoxycytidine) inhibit or delay the development of oral cavity SCC (squamous cell carcinoma). This is the first demonstration of a positive cancer preventive effect of 5-aza-dCdR (epigenetic therapy) in an oral cavity carcinogenesis model. Again, with funding from this grant, we have developed a knockout mouse model (LRAT null mice) in which vitamin A (retinol) deficiency can be generated quickly and easily. We have used these mice to establish that retinol deficiency increases the incidence of oral SCC. In Aim 1, we will use a pharmacological approach to determine if retinoid and/or DNMTase inhibitors inhibit cancer progression in the 4-NQO model and to show how this inhibition occurs by measuring the expression of key molecular markers that are associated with cancer development. Specifically, we will test an improved dosing schedule of RA with 5-aza-dCdR. We will also test the RXR selective retinoids bexarotene (LG1069) and LG100268, which have achieved positive results in other cancer chemoprevention models. We will test zebularine, a potent inhibitor of DNMTases, which exhibits excellent drug stability and has shown positive results in other cancer prevention models. We will measure numbers and severity of oral cancer lesions and various molecular markers known to change in human SCCHN, e.g. p16, cyclin D1, cyclooxygenase-2, EGFR, phosphoSTAT3, and cell proliferation (BrdU or PCNA). We will also examine proteins that effect epigenetic changes, e.g. JMJD3. In Aim 2, by using a transgenic strategy, we will permanently mark cells in the basal layer of the oral epithelium. We will test the hypothesis that these cells give rise to cancer, and use molecular markers to characterize these cells during the oral carcinogenesis process. To examine the effects of cancer chemopreventive agents at the cellular level, we will also follow the permanently marked cells in 4-NQO treated mice that are then treated with the drugs described in Aim 1. To gain information about the mechanism(s) by which retinol deficiency leads to a higher incidence of oral SCCs, we will perform similar cell marking experiments by crossing K14/creER(Tam);LRAT null mice with Rosa(lacZ);LRAT mice. From this research we will understand more about the mechanisms by which these dietary and chemotherapeutic approaches can inhibit the development of oral SCC, develop rational approaches for oral cancer prevention, and identify the cells involved in the carcinogenesis process that are influenced by cancer chemopreventive drugs. Many cancer chemoprevention trials in humans have not been successful, pointing to the need to carry out more fundamental studies and to test the idea that combination therapy may be essential for successful chemoprevention.